JP2020529606A - Lateral flow analysis and equipment for application to skin care - Google Patents

Abstract

The present invention relates to a diagnostic kit and method based on a lateral flow analyzer that detects the presence or absence of one or more test analytes in a test sample taken from mammalian skin, or detects the amount thereof. [Selection diagram] Fig. 3

Description

The present invention relates to a diagnostic kit and method based on a lateral flow analyzer that detects or detects the presence or absence of one or more test analytes in a test sample taken from mammalian skin.

The rapid development of genomics, transcriptomics, proteomics, and regulomics has enabled large-scale analysis of molecular and cellular mechanisms. One of the key outcomes of these studies is the development of functional genomics, where cells from other individuals make significant differences in genomic structure, gene and protein expression profiles and regulatory mechanisms that control specific cell functions. It is an advanced understanding of having. This has raised interest in detecting and / or quantifying biomarkers that can assess the current state of mammals through the presence, absence and / or concentration of one or more biomarkers.

There is also a need to assess how effective individual-level treatments are in the areas of personalized medicine and personalized skin care.

In the context of personalized skin care, the wrinkle-removing and anti-aging effects of cosmetics are generally based on the assumption that cosmetics have similar effects on all individuals. But this is not true. Different people and different skin types react differently to cosmetics. Therefore, there is a need for a point care device that can determine the personal impact and individual reaction to a particular type of skin care product.

Skin quality depends on biological processes that control and regulate skin tissue, skin structure and skin function. The basic biological mechanisms underlying skin performance are associated with maintaining, renewing, and functioning different cell populations within the skin. For example, extracellular matrix cutaneous fibroblast control homeostasis, skin keratin-producing cell control barriers, immune cells and various factors contribute to stimulating processes and the fight against infections. The functional networks (molecular mechanisms) that control these processes are relatively well known, and there are key players in these networks. The levels and activities of different cytokines and growth factors control the balance of cellular processes in the skin, such as different cell population proliferation and differentiation, extracellular matrix synthesis and degradation, and metabolic activity. The combination of these activities leads to skin quality and aesthetic appearance.

Interleukin levels can be used to determine skin condition and also provide recommendations on how to improve skin quality (appearance, function, structure).

One challenge faced by lateral flow analysis methods is to provide samples to be tested, in particular to provide skin samples, and in particular to to provide reproducible and / or constant skin samples. Is to provide.

WO2014184151 A1 discloses a point care diagnostic device that can analyze without invading pervasive factors hidden from the skin surface based on lateral flow analysis technology.

US2005 / 0175992 discloses a method for rapidly diagnosing targets in human body fluids. In particular, a lateral flow analysis method is used in which a cotton member is used to take a non-invasive sample from the eye fluid.

Therefore, there is a need for kits and methods for collecting and analyzing analytes from the skin, especially for point care devices that can be detected quickly. There is also a demand for sampling methods for point care devices that can provide samples reproducibly and / or consistently as compared to prior art.

The present invention has been made in view of the above prior art, and an object of the present invention is to detect, or to detect, whether or not one or more test analytes are present in a test sample taken from mammalian skin. It is to provide a diagnostic kit for detecting the amount.

One aspect of the present invention is a diagnostic kit for detecting the presence or absence of one or more test analytes in a test sample taken from the skin surface of a mammal, or detecting the amount thereof. ,
a) Separation cotton materials 200, 301 used when collecting the test sample, the cotton material has sample collection pads 201, 101 attached to the support member 202, and
b) A lateral flow analyzer configured to accept and hold the separated cotton material,
It is to provide a diagnostic kit consisting of.

In addition, the inventor provides a modified lateral flow analysis that analyzes IL-1a, IL-1RA, IL-8 levels, etc. in the skin. Therefore, the second aspect of the present invention is a method of detecting the presence or quantity of one or more test analytes, which method is:
a) A step of providing a separation cotton material 202 with a sample collection pad 201 as defined herein, the sample pad having a test sample taken from the skin surface of a subject using the separation cotton material.
b) A step of inserting a cotton material consisting of a sample collection pad having the test sample into a lateral flow analyzer configured to receive the separated cotton material.
c) Steps to proceed with lateral flow analysis,
Is to provide a method consisting of.

1a-1d are perspective views of different embodiments of the porous support assembly 100 (also referred to as side flow analysis stripes) according to the present invention. In FIG. 1a, a sample pad 101, a bonding pad 102, a detection zone 105 and an indicator zone (106) (these two zones are fixed to the porous support 107), a suction pad 104 and a backing material 108 are shown. The porous support assembly 100 having is shown. L indicates the lateral flow direction, and the region DA is the detection region. The sample pad 101 shows a porous support assembly that is separated from the remaining porous support assembly. FIG. 1a shows another embodiment, which has a sample pad 101, a joining pad 102, a detection zone 105, and an indicator zone 106 on the porous support portion 107, and the suction pads 104 are adjacent to each other or overlap each other. , They are placed on the backing material 108. Shown in FIG. 1c with lateral flow stripes, the sample pad 101 is separated from the remaining porous support assembly.

An embodiment of the present invention is shown, showing a separated cotton material 200 having a support member 202 having an opening 205 formed at a terminal portion 204. The case where the separation cotton material 200 does not have the sample collection pad 201 and the case where the separation cotton material 200 has the sample collection pad 201 which is attached to cover the peripheral portion of the opening are disclosed. The support member 202 is formed with a notch 206 at one end of the support member 202. The notch 206 is configured to interact with the curved portion 303 of the lateral flow device 300 to orient the position of the cotton material at the insertion position in the lateral flow device 300 to ensure that position. .. Further, FIG. 2 discloses an example of a cotton material 200 in which the width of the near end portion 203 of the support member 202 is widened in order to form a finger grip (handle).

In one embodiment of the present invention, a lateral flow device 300 having a separation cotton material 200 to be inserted into a sample pad slot 306 is shown, and the slot 306 is configured to react with a notch 206 of the separation cotton material 200. The separated cotton material 200 is oriented and positioned in the lateral flow device 300 by having the curved portion 303. The lateral flow device 300 is provided with a holding member 302 that is closed at the upper part of the separation cotton material 200 to hold and lock the cotton material at the insertion position of the side flow device 300. The holding member 302 is provided with an opening 304 that is aligned with the sample collection pad 201 when the holding member is in the locked position, and the sample collection pad is exposed through the opening 304. The installed running buffer is accessible. Further, the lateral flow device 300 has a reaction window 305 capable of visually observing the detection region DA.

The results of in vitro tests of various substances for sampling are shown. As a test sample, 80 μl of a standard protein solution (2 ng / ml IL8, 4 ng / ml IL1A, 8 ng / ml IL1RA recombinant protein) was used. The signal strength was measured as mV.

It is the result of an in vivo test of the material selected for sampling. FIG. 5A shows the forehead and FIG. 5B shows the inside of the arm. The signal strength was measured as mV.

The results of comparing the blocked sample pad C083 on the inside of the forearm skin with the corresponding unblocked sample pad are shown. The signal strength was measured as mV.

The results of comparing the sampling procedures (in vivo) are shown. FIG. 7A is the forehead and FIG. 7B is the cheek. There are 3 applicants (JA, AL, AS). The signal strength was measured as mV.

This is the result of comparing different in vivo wiping procedures for two applicants (JA, AL). Z-shaped movement vs. 5 cm ² area for 5 seconds. The signal strength was measured as mV.

It shows the effect of pretreatment of the skin before taking a sample and how long the pretreatment can last. FIG. 9A shows pretreatment with water, FIG. 9B shows pretreatment with EtOH, and FIG. 9c shows pretreatment of skin by wiping with a cosmetic wipe. The light gray bar is 1L1A and the dark gray bar is IL1RA. The signal strength was measured as mV.

Those skilled in the art will appreciate that certain features of FIGS. 1-5 do not necessarily have to be created on a constant scale given the benefits of disclosure herein. To aid in the understanding of the exemplary embodiments disclosed herein, the dimensions and features of the drawings can be enlarged, distorted and modified as compared to other drawings.

Those skilled in the art will appreciate that the features of the drawings can be replaced with others to obtain further embodiments.

Specific terms are used for clarity when describing examples of the present invention. However, the present invention should not be limited to the particular terms thus selected, and it is understood that each particular term includes all technical equivalents that act similarly to achieve similar objectives. Will be done.

A first aspect of the present invention is to provide a diagnostic kit for detecting the presence or absence of one or more test analytes in a test sample taken from mammalian skin, or for detecting the amount thereof. Diagnostic kit
a) Separation cotton materials 200, 301 with sample collection pads 201, 101 attached to support member 202, configured to be used to collect the test sample.
b) Lateral flow analyzer 300, configured to accept and hold the separated cotton material,
It is composed of.

The diagnostic kit according to the present invention shall be used in a point-of-care device that uses the separated cotton material 200 of the diagnostic kit to detect the presence of one or more test analytes in a test sample taken from the skin. Can be done.

The isolated cotton material of the diagnostic kit according to the present invention is constructed in a form suitable for taking a test sample from the skin surface of a mammal. In a preferred embodiment, the mammal is a human. The cotton materials 200 and 301 have a support member 202, and the sample collection pads 201 and 101 are attached to the support member 202, preferably on one side of the support member. Generally, the support member 202 is used when collecting a sample from the skin, for example, when the sample collection pads 201, 101 are placed on the skin and the pads are moved while being controlled on the skin by using the support member 202. , Used as a handle.

In one embodiment, the support member is elongated, for example, 2.5 times, 3 times, at least 4 times, etc., at least twice the width of the member. In one embodiment, the support member has the near end 203 formed as a finger grip, and the sample collection pads 201 and 101 attached to the opposite end 204. The shape of the near end portion 203 is formed so that the support member 202 can be stably grasped between two or more fingers. FIG. 2 discloses an example in which the width of the near end portion 203 of the support member 202 is widened in order to grip the support member 202 well with a finger. As described above, in one embodiment, the width of the near end portion 203 of the support member is wider than that of the end portion 204. In one embodiment, the area and shape of the near end 203 of the support member correspond to the thumb of an adult human, which allows the support member to be stably gripped.

In one embodiment, the support member 202 is flexible along the vertical axis of the support member. Since the support member 202 is made of a flexible material, the support member 202 bends lightly when the sample collection pads 201 and 101 are pressed against the skin and the support member 202 is evenly moved on the skin to collect the sample material. ing. Since the support member 202 is flexible, the risk of damaging the skin is reduced. In a preferred embodiment, the support member 202 is made of a plastic material, for example, the support member 202 is a plastic material having a thickness of 1 mm or less, between 2 mm and 0.5 mm, between 2 mm and 1 mm, and thinner than about 2 mm. It is made of, which makes it flexible along the vertical axis of the support member 202.

In a preferred embodiment according to the present invention, an opening 205 is formed at the end 204 of the support member 202, and the sample collection pads 201 and 101 are configured to cover the opening. In this embodiment, the sample collection pads 201 and 101 attached to the support member 202 cover the opening and its periphery. In one embodiment, the sample collection pads 201, 101 are attached to the support member 202, and the sample collection pads cover the opening 205.

The sample collection pads 201 and 101 can be attached to the support portion 202 near the periphery of the opening. The sample collection pads 201 and 101 can be attached to the support member 202 apart from the periphery of the opening. Generally, the sample collection pads 201, 101 are attached to one side of the support member 202. In one embodiment, the area of the opening 205 corresponds to at least 50 percent, such as at least 60 percent of the sample collection pad, and at least 70 percent, eg, at least 70 percent, and, eg, at least 90 percent. Etc., corresponding to at least 80%, and for example, 95%.

The sample collection pads 201, 101 of the cotton material 200 form part of the porous support assembly 100 when inserted into the lateral flow device 300, i.e. the sample collection pads come into contact with other elements of the assembly. To do. By adding a running buffer to promote lateral flow within the porous support assembly, the openings can approach the sample collection pads 201, 101. In the present invention, the running buffer may be any liquid buffer, such as a PBS buffer, as long as it is suitable for promoting lateral flow in the porous support assembly.

The sample collection pads 201, 101 take a test sample from the skin and then combine with the porous support assembly 100 to form part of the porous support assembly 100 and remove the test sample from the porous support assembly 100. Made of materials suitable for. In one embodiment, the sample pads 201, 101 are made of a cellulosic material or a cellulose derivative such as nitrocellulose, polyether sulfone, polyethylene, nylon polyvinylidene fluoride (PVDF), polyester, polypropylene, glass fiber, cotton, cloth, etc. There is. In a preferred embodiment, the sample pads 201 and 101 are made of a cellulosic material such as nitrocellulose or a cellulose derivative.

The sample pads 201 and 101 are in the form of a sheet or the like. In one embodiment, the sample collection pads 201, 101 are in the form of a layer of one or more sheets, such as a layer of two sheets.

The average thickness of the sample pads 201, 101 is preferably less than 2 mm, such as 1 to 0.80 mm, preferably less than 1 mm, such as less than 0.95 mm, for example 0.85 to 0.80 mm, such as 0.83 mm. Less than mm. In one embodiment, the sample pads 201 and 101 are layers composed of two sheets, and the thickness of each sheet is less than 0.50 mm, such as 0.49 mm to 0.40 mm.

To prevent and reduce the bias due to absorption of one or more test analytes or the bias when removing the one or more test analytes from the sample collection pads 201, 201, use a blocking buffer in the sample collection pad. 201 and 101 can be pre-wet. In one example, this blocking buffer is a PBS buffer consisting of 1% BSA, or a buffer consisting of 10 mM borate, 3% BSA, 1% PVP-40, 0.25% Triton X100 pH 8.0.

In a particular embodiment, the sample collection pads 201, 101 are in the form of a cellulosic material or a cellulose derivative, such as nitrocellulose, pretreated with a blocking buffer, the thickness of the sample collection pads 201, 101 being It is 0.85 to 0.80 mm, such as 0.83 mm.

In a preferred embodiment of the present invention, the support member 202 of the separation cotton material 200 is provided with a notch 206 on the end portion 204 of the support member 202 or in the vicinity thereof. The lateral flow device 300 fits snugly with the notch 206 on the support member 202 and has a curved portion 303 that directs and positions the end portion 204 of the support member 202 when the cotton material is inserted into the side flow device 300. are doing. As described above, the curved portion and the notch are configured such that the cotton material, particularly the sample collection pads 201 and 101, is correctly and surely oriented and positioned in the lateral flow device 300. As described above, the curved portion and the notch are formed by inserting the cotton material, particularly the sample collection pads 201 and 101, into the lateral flow device 300, and the sample collection pads 201 and 101 of the cotton material 200 are one of the porous support assemblies 100. The part, i.e., the sample collection pad, is configured to ensure contact with other elements of the assembly. Therefore, in one embodiment, the separating cotton material 200 has a notch 206 on or near the end of the end 204 of the support member 202, and the lateral flow device 300 has a notch 206 in the support member. It has a curved part that fits snugly with. As a result, the sample collection pads 201, 101 of the cotton material 200 form part of the porous support assembly 100 when inserted into the lateral flow device 300. That is, the sample collection pad comes into contact with other elements of the assembly.

In another embodiment of the present invention, the lateral flow device 300 has an opening 304, the opening is aligned with the opening 205 of the cotton material, and the cotton material becomes a lateral flow device. Upon insertion, the sample collection pads 201, 101 are exposed through the opening 304. The sample collection pads 201 and 101 can be pre-wet with a running buffer, but when the separation cotton material 100 is inserted in the lateral flow device 300, a running buffer can be further added to the opening.

The combined use of bends and cuts and the addition of running buffers to the sample collection pads 201, 101 ensure that the sample collection pads 201, 101 line up correctly with respect to the openings.

In one embodiment, the lateral flow device is configured to accept the end of the cotton material to ensure the position of the sample collection pads 201, 101, 301 within the lateral flow device. Has 306.

In another embodiment, the lateral flow device has a holding member 302 that holds the end of the cotton material and ensures the position of the end of the cotton material that constitutes the sample collection pads 201, 101. In yet another embodiment, the holding member can be attached to the body of the lateral flow device by a hinge. In the open state, the end 204 of the support member 202 constituting the sample collection pads 201, 101, 301 can be inserted into the device, and there is a sample pad slot 306 configured to receive the end of the cotton material. Further facilitates insertion. In the closed state, the holding member is closed around the sample collection pads 201, 101, 301, whereby the sample collection pads 201, 101, 301 are stably held in the lateral flow device 300. In one embodiment, the holding member 302 closes the end of the cotton material and is locked to the body of the lateral flow device. Preferably, the holding member 302 of the device is used in combination with a bend and a notch so that the cotton material, and especially the sample collection pads 201, 101, are correctly and reliably oriented and positioned within the lateral flow device. ..

In a preferred embodiment, the holding member 302 has an opening 304 configured to line up with the cotton opening 205, and when the cotton material is inserted into the lateral flow device, the opening 304 is opened. The sample collection pads 201 and 101 are exposed through the sample collection pads 201 and 101. The opening 304 can have the shape of a port, such as a conical port with a base that is wide upwards and narrow downwards. In this configuration, lateral flow can be promoted within the porous support assembly 100 by further adding a running buffer to the sample collection pads 201, 101.

In the specification of the present invention, "lateral flow" refers to a flow of a liquid, which is dissolved or dispersed in the liquid together with the liquid (including a test analysis product) in the flow of the liquid. The element moves laterally via a porous support assembly (100, referred to as a capillary bed or lateral fluidized stripe). This is subject to the condition that the element is not permanently trapped and that it is not moved in the liquid by other means. Such an analysis that relies on lateral flow is called lateral flow analysis. Preferably, when the porous support assembly is made of non-moisture absorbent, the elements in the liquid move at essentially equal rates through the capillary bed. If the porous support assembly is made of a moisture absorber, this material can affect the movement of one or more elements. If the porous support assembly is composed of or has a moisture absorber, the properties of the porous support assembly may be changed to be the same as or essentially the same as the non-moisture absorber. The material can be treated with a blocking agent such as PBS buffer consisting of BSA and / or Triton X-100.

The lateral flow analysis is based on a porous support assembly 100 capable of transferring fluid by the action of capillary force, i.e., a capillary bed (porous paper, polymer sintered body, etc.). The porous support assembly 100 is a collection of porous support elements, and when fluid (such as a running buffer) is added to the assembly, the elements are in contact with each other. One of the porous support elements of the porous support assembly 100 is the sample collection pads 101, 200 that become part of the porous support assembly 100 when the cotton material is in the insertion position of the lateral flow device. The porous support assembly 100 is also referred to as a lateral analysis stripe.

In one embodiment of the present invention, when the lateral flow device 300 is combined with a sample collection pad attached to a cotton material (at which time, the sample collection pad is composed of an elution zone 101, a junction zone 102 and a detection region DA). The lateral flow device is configured to form the porous support assembly 100.

The joining zone can be integrated with the relatively large porous element of the porous support assembly 100, such as the porous support stripe 107. The joining zone can be in the form of one element of the porous support assembly 100. In a preferred embodiment, the bonding zone is in the form of a bonding pad 102.

The sample collection pads 101 and 200 function as sponges and hold test samples. Once the sample collection pad gets wet, the test sample containing one or more test analytes moves from the sample collection pads 101, 200 into the adjacent elements of the porous support assembly 100. The interphase between the sample collection pads 101, 200 and the adjacent elements of the porous support assembly is called the elution zone. Adjacent elements of the porous support assembly are generally bond zones, preferably in the form of bond pads 102. The binding zone / binding pad 102 is generally one or more indicator affinity molecules, such as affinity molecules tagged with a detection probe designed to constrain one or more test analytes within the test sample. Includes. The test sample and one or more affinity molecules are mixed, and one or more affinity molecules having an affinity for one or more test analytes in the test sample further include detection zone 105 and indicator zone 106 (both). Move to the detection region DA, which may contain one or more stripes), and another set of one or more affinity molecules bind to each other while being immobilized. From the binding pad, one or more analytes in the test sample bind to the affinity molecule from the binding pad by the time the test sample mixed with the affinity molecule reaches the detection region DA. The complex is then constrained, in turn, by the affinity molecules of the stripes within the detection region 105. After a while, as more liquid passes through the detection zone, the detection probe accumulates and the stripes change color. The detection probe can be, for example, gold particles or latex particles that bind to the affinity molecule, which allows the affinity molecule tagged with the detection probe to be prepared. The detection area DA may consist of an indicator zone 106 that is controlled to demonstrate that the lateral flow analysis was performed accurately. The indicator zone 106 may consist of one or more stripes with affinity molecules that only bind from the binding pad to the affinity molecule tagged with the detection probe and do not move. On the other hand, the affinity molecule in the detection zone 105 binds to the complex between the analyte and the indicator affinity molecule, such as the affinity molecule tagged with the detection probe from the binding pad. After passing through the detection region DA, the fluid generally enters the suction pad 104, which receives the fluid as it travels through the entire porous support assembly 100. Thus, in one example, the detection region DA is a detection zone 105 containing one or more affinity molecules that selectively hold one or more test analytes, and optionally one or more indicator affinity molecules. Consists of an indicator zone 106 containing one or more affinity molecules that selectively retain.

The detection zone 105 can be arranged upstream or downstream of the indicator zone 106. Detection zone lines and stripes can be placed substantially perpendicular to the flow of test samples. In some embodiments, the lines can be oriented substantially parallel to the flow of the test sample. The lines and stripes of the detection zone 105 and the indicator zone 106 do not necessarily have to be lines or stripes, and may be other shapes such as dots or patterns.

In one embodiment, the lateral flow device further has a suction pad 104. The suction pad 104 is part of the porous support assembly 100 and helps to promote capillary action and promote fluid flow from the sample pad 101, the coupling pad 102 through the detection region DA.

In another embodiment, the lateral flow device has a backing material 108 on the back side of the porous support assembly 100 so as to face away from the elution zone. Since the backing material 108 does not permeate the liquid, the fluid flowing through the porous support assembly 100 does not leak through the backing material 108. Suitable materials for support include, but are not limited to, glass and polymeric materials such as polystyrene, polypropylene, polyester, polybutadiene, polyvinyl chloride, polyamide, polycarbonate, epoxides, methacrylic hydrochloride, and polymelamine.

The porosity support assembly 100 is a combination of one or more porosity elements and two or more porosity elements such as sample collection pads 201, 101. Here, the cotton material 200 constituting the sample collection pad is inserted into the lateral flow device 300. The element is preferably in the form of a membrane, such as a sheet-like membrane. The porous support assembly 100 has a thickness of 4 mm or less, such as 4 mm, 3 mm, 2 mm, 1 mm, etc., and the width and length are larger than the thickness. In some embodiments, the width and length of the porous support assembly 100 is more than the thickness (eg, 3,4,5,6,7,8,9,10,50 times or 4,5,6,7,8). , 9,10,50 times larger). In some embodiments, the porous support assembly 100 is a quadrangle, such as a rectangle, square, etc., and in some embodiments, the porous support assembly 100 is circular. When the porous support assembly 100 has an irregular shape that is not square or rectangular, its width, length and thickness apply to the maximum value of the irregular shape. For example, the width of a circle is the diameter. Examples of width and length are 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 11mm, 12m, 13mm, 14mm, 15mm, 16mm, 17mm, 18mm, 19mm, 20mm, 21mm such as 5 ~ 30mm. , 22mm, 23mm, 24mm, 25mm, 26mm, 27mm, 28mm, 29mm, 30mm, 35mm, 40mm.

Thus, in one embodiment, the porous support assembly 100 of the lateral flow device 300 has an average thickness of 4 mm or less and a width and length greater than the thickness. Here, the lateral flow device is configured to have a lateral flow direction (L) in the direction of the plane created by the width and length of the porous support assembly.

In one embodiment, the lateral flow device has a reaction 305 configured to visually detect the detection area DA.

The diagnostic kit of the present invention can be used for test analytes that are present on the skin and can be collected using the separated cotton material 200. In one example, the one or more test analytes were chemokines, interleukins, growth factors, hormones, enzymes and IL-1a, IL-1b, IL-1RA, IL-8, CCL-2, CCL-5. , CCL-27, CXCL-1, CXCL-2, CXCL-9, Trappin2 / Elafin, hBD-1, hBD-2, VEGF and TSLP and other molecules present on mammalian skin selected from the list. Selected from a list of. In a preferred embodiment, the test analytes are IL-8, IL-1a, IL-1RA.

As described above, the diagnostic kit of the present invention can be used in a point care device that detects the presence or absence of one or more test analytes in a test sample taken from the skin using the separate cotton material 200 of the diagnostic kit. You can. The display can be done visually. That is, the presence or absence of one or more colored test lines, which is also called a test stripe in the detection zone 105, is visually determined, and the confirmation / verification of the test is performed by the presence of one or more colored indicator lines / stripes in the indicator zone 106. / Or can be done by absence. The tests are qualitative (presence or absence) and quantitative, detection / quantification can be assisted by a reading device, or can be purely detected visually by the user of the lateral flow analysis.

One aspect of the present invention is a method of detecting the presence or amount of one or more test analytes, which method is:
a) A step of providing a separate cotton material 200 having a sample collection pad 201 as defined herein, wherein the sample pad has a test sample taken from a subject skin surface using the separate cotton material. And
b) A step of inserting a cotton material consisting of a sample collection pad containing the test sample into a lateral flow device that accepts the separated cotton material.
c) Steps to proceed with lateral flow analysis,
Consists of.

Another aspect of the invention is a method of detecting the presence or quantity of one or more test analytes.
a) Using a cotton material 200 made of sample collection pad 201, a test sample was taken from the skin surface of the subject.
b) A step of inserting a cotton material consisting of a sample collection pad containing the test sample into a lateral flow analyzer configured to receive the separated cotton material.
c) Steps to proceed with lateral flow analysis,
Consists of.

The subject is a mammal, but preferably a human. A test sample is taken by applying the separated cotton material 200 having the sample collection pad 201 to the skin of a mammal, preferably a human. The skin area can be, for example, the forehead, cheeks, the inside of the arm, or the part of the arm that is routinely exposed to the sun. Apply the separated cotton material to a predetermined amount of area, such as a range not exceeding 5 cm ² . The separated cotton material can be applied for a predetermined time such as 5 seconds or 30 seconds. The test sample can be collected by moving the cotton material in a predetermined movement, such as moving the cotton material in a Z shape on the skin.

Collection can be assisted by wetting the sample collection pad 201 with a certain amount of fluid. In a preferred embodiment, the sample collection pad 201 of the separated cotton material 200 is prewet with a buffer such as a predetermined amount of buffer before sampling. This buffer may be any appropriate buffer such as a PBS buffer. The buffer used to prewet the sample collection pad may be the same as the buffer used as the running buffer in the steps of the lateral flow analysis step.

In another preferred embodiment, a running buffer is added to the sample collection pad inserted into the lateral flow analyzer. When a running buffer is added to the sample collection pad inserted into the lateral flow analyzer, sufficient fluid flows laterally or is promoted within the porous support assembly 100. If the lateral flow device 300 has an opening 304, the opening can be used as a port when adding a running buffer to the sample collection pad 201 inserted in the device.

In one embodiment, the lateral flow device is composed of an elution zone 101 and a detection region DA. Here, the sample collection pad is the elution zone 101.

In another embodiment, when the porous support assembly 100 is combined with the sample collection pad attached to the cotton material, the lateral flow device 300 combined with the sample collection pad is from the elution zone 101, the coupling zone 102 and the detection region DA. The lateral flow device is configured to form the porous support assembly 100.

In one embodiment, the porous support assembly 100 has a thickness of 4 mm or less, and the width and length are greater than the thickness. Here, the lateral flow device is configured to have a lateral flow direction L, which is substantially the direction of the plane created by the width and length of the porous support assembly 100.

In another embodiment, the detection region DA selectively selectively holds one or more affinity molecules having one or more affinity molecules and optionally one or more indicator affinity molecules that selectively hold one or more test analytes. It is composed of an indicator zone 106 having one or more affinity molecules retained in.

In general, the present invention is directed to diagnostic kits that provide an integrated system that collects and detects the presence or absence of one or more test analytes in a test sample taken from the skin in the widest possible range. In some embodiments, the amount of one or more test analytes is also detected in the quantitative analysis. The diagnostic kit uses a lateral flow device 300, separate cotton materials 200, 301 and one or more reagents that detect one or more test analytes in the test sample. The analytical reagent has an affinity molecule tagged with a detection probe capable of producing a detection signal indicating the presence or absence of one or more test analytes in the test sample. One method for determining the amount of one or more test analytes is to prepare an appropriate standard curve using the known concentrations of one or more test analytes.

One or more analytes analyzed using the methods of the invention include chemokines, interleukins, growth factors, hormones, enzymes and IL-1a, IL-1b, IL-1RA, IL-8, CCL-2, CCL. Others present on mammalian skin as selected from the list consisting of -5, CCL-27, CXCL-1, CXCL-2, CXCL-9, Trappin2 / Elafin, hBD-1, hBD-2, VEGF and TSLP. You can choose from a list of molecules. In a preferred embodiment, the test analyte is a combination of IL-8, IL-1a, IL-1RA.

If necessary, an appropriate reading device, such as an optical reading device, can be used in certain embodiments to measure the strength of the probe. In general, the shape and configuration of an optical reader will vary greatly depending on the probe to be measured. For example, the optical detection techniques that can be used are, but are not limited to, light emission (ie, fluorescence, phosphorescence, etc.), absorption (ie, fluorescent or non-fluorescent), diffraction, and the like. According to the present invention, the presence or concentration of an analyte can be qualitatively, quantitatively or semi-quantitatively determined. For example, the intensity of the signal produced by the detection probe constrained in the detection zone 105 or the indicator zone 106 can be used to quantitatively or semi-quantitatively determine the amount of the analyte.

In a preferred embodiment, an image pickup device such as a mobile phone having a camera is used to capture an image of the detection region DA. The image is then transmitted to a computer system consisting of an image processor and a database (eg, a remotely located server) to retrieve the image features and compare the features with the corresponding features recorded in the database. Analyze the image by doing so. This computer system generates output data based on the image features, returns the data to the mobile phone used to capture the image, and is transmitted to the user.

As described above, in one embodiment of the present invention, the method of the present invention further includes d) an image of the detection region DA and a step of transmitting the image to a computer system including an image processing device and a database. An image feature is extracted by an image processing device, the image feature is stored in a database, and the computer system generates at least one output data based on the image feature. In another embodiment, the image is captured using a mobile device, such as a mobile phone configured to capture the image. In yet another embodiment, the output data generated by the computer system is transmitted to the mobile device.

In describing the examples of the present invention, the combinations and substitutions of all the examples are not explicitly described. Nevertheless, the fact that certain measures are listed in different dependent claims and described in different embodiments does not mean that the combination of these means is not being used effectively. The present invention includes all possible combinations and substitutions of the described examples.

The inventor believes that "having" as used herein can be interchanged with the term "consisting of" in all cases.

The present invention is further described by the following non-limiting items.

Item 1. A diagnostic kit that detects the presence or absence of one or more test analytes in a test sample taken from the skin surface of a mammal, or detects the amount thereof.
a) Separation cotton materials 200, 301 configured to be used in collecting the test sample, the cotton material having sample collection pads 201, 101 attached to the support member 202.
b) Consists of a lateral flow analyzer 300, which is configured to receive and hold the separated cotton material.

Item 2. The diagnostic kit of item 1, the lateral flow analyzer 300 is composed of one or more porous elements, and the sample collection pads 201, 101 have cotton materials 200, 301 inserted into the lateral flow analyzer 300. When so, it is configured to form part of the porous support assembly 100.

Item 3. A diagnostic kit for item 1 or 2, wherein the support member is configured to have a near end 203 configured as a finger grip and an end 204 to which the sample collection pad 201 is attached.

Item 4. Diagnostic kits for items 1-3, the support member 202 being flexible along the longitudinal axis of the support member.

Item 5. A diagnostic kit for items 1-4, the end 204 of the support member 202 has an opening 205 configured to be covered by a sample collection pad 201.

Item 6. Diagnostic kits for items 1-5, the sample collection pads 201, 101 are attached to a support member 202 such that the sample collection pads cover the opening 205.

Item 7. Diagnostic kits for items 1-6, wherein the area of the opening 205 corresponds to at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sample collection pad. To do.

Item 8. Diagnostic kits for items 1-7, sample collection pads 201, 101 are cellulosic materials such as nitrocellulose, polyether sulfone, polyethylene, nylon polyvinylidene fluoride (PVDF), polyester, polypropylene, glass fiber, cotton, cloth, etc. And made of cellulose derivatives.

Item 9. Diagnostic kit for items 1-8, the sample collection pads 201, 101, PBS buffer consisting of 1% BSA, or 10 mM borate, 3% BSA, 1% PVP-40, 0.25% Triton X100 pH8 Preprocess with a blocking buffer such as .0.

Item 10. It is a diagnostic kit of items 1 to 9, and the sample collection pads 201 and 101 have the shape of a sheet or the like.

Item 11. Diagnostic kits for items 1-10, said sample collection pads 201, 101 having the shape of a layer of one or more sheets, such as a layer of two sheets.

Item 12. It is a diagnostic kit of items 1 to 11, and the thickness of the sample collection pads 201 and 101 is less than 2 mm such as 1 to 0.80 mm, preferably less than 1 mm such as less than 0.95 mm, for example 0.85 such as 0.85 to 0.80 mm. It is less than mm.

Item 13. The diagnostic kits of items 1 to 12, the sample collection pads 201 and 101 have the shape of a layer composed of two sheets, and the thickness of each sheet is less than 0.5 mm such as 0.49 to 0.40 mm.

Item 14. Diagnostic kits for items 1 to 13, sample collection pads 201 and 101 are cellulosic materials and cellulose derivatives such as nitrocellulose pretreated with a blocking buffer, and sample collection pads 201 and 101 have a thickness of 0.85 to 0.80. mm.

Item 15. It is a diagnostic kit of items 1 to 14, and the support member is made of a plastic material.

Item 16. Diagnostic kits for items 1-15, the support member 202 is made of a flexible material, and sample collection pads 201, 101 are pressed against the skin and moved over the skin to collect test samples. Then, the support member 202 is slightly bent.

Item 17. It is a diagnostic kit of items 1 to 16, and the support member is made of a plastic material, and the thickness thereof is less than about 2 mm, such as 1 mm or less, between 2 mm and 0.5 mm, between 2 mm and 1 mm, and the like.

Item 18. Diagnostic kits for items 1-17, one end of the end 204 of the support member 202 has a notch 206, the lateral flow device when the cotton material is inserted into the lateral flow device. The curved portion 303 is configured to direct and position the end portion of the support member.

Item 19. Diagnostic kits for items 1-18, the lateral flow device having an opening 304 configured to line up with the cotton material opening, a sample collection when the cotton material is inserted into the lateral flow device. The pads 201 and 101 are exposed through the opening 304.

Item 20. Diagnostic kits for items 1-19, where the lateral flow device has sample pad slots 306 configured to accept the end of the cotton material, thereby the sample collection pads 201, 101, in the lateral flow device. Secure the position of 301.

Item 21. Diagnostic kits for items 1-20, the lateral flow device is configured to hold the end of the cotton and ensure the location of the end of the cotton consisting of sample collection pads 201, 101. It has a holding member 302.

Item 22. It is a diagnostic kit of items 1 to 21, and the holding member is attached to the main body of the lateral flow device by a hinge.

Item 23. A diagnostic kit for items 1-22, the holding member 302 is folded over the end of the cotton material and locked to the body of the lateral flow device.

Item 24. A diagnostic kit for items 1-23, the holding member 302 has an opening 304.

Item 25. Diagnostic kits for items 1-24, the opening 304 has the shape of a port, such as a conical port with a base that is wide upwards and narrow downwards.

Item 26. Diagnostic kits for items 1-25, mammals are humans.

Item 27. Diagnostic kits for items 1-26, the lateral flow device is configured to form a porous support assembly 100 when combined with a sample collection pad attached to cotton material, at which time the sample collection pad. The side flow device 300 combined with the above is composed of an elution zone 101, a coupling zone 102, and a detection region DA.

Item 28. Diagnostic kits for items 1-27, the binding zone having the shape of a binding pad 102.

Item 29. Diagnostic kits for items 1-28, the lateral flow device further comprising a suction pad 104.

Item 30. Diagnostic kits for items 1-29, the lateral flow device has a backing material 108 on the back side of the porous support assembly 100 so as to face away from the elution zone.

Item 31. Diagnostic kits for items 1-30, the average thickness of the porous support assembly is 4 mm or less, and the width and length are greater than the thickness. In this case, the lateral flow device is configured to have a lateral flow direction L in the direction of the surface created by the width and length of the porous support assembly 100.

Item 32. Diagnostic kits for items 1-31, the detection region DA includes a detection zone 105 containing one or more affinity molecules that selectively hold one or more test analytes, and optionally one or more indicator affinities. It is composed of an indicator zone 106 containing one or more affinity molecules that selectively hold the molecule.

Item 33. Diagnostic kits for items 1-32, the lateral flow device has a reaction window 305 capable of visually observing the detection area DA.

Item 34. Diagnostic kits for items 1-3, said one or more test analytes are chemokines, interleukins, growth factors, hormones, enzymes and IL-1a, IL-1b, IL-1RA, IL-8, CCL- 2, CCL-5, CCL-27, CXCL-1, CXCL-2, CXCL-9, Trappin2 / Elafin, hBD-1, hBD-2, VEGF and TSLP on mammalian skin, as selected from the list. Selected from a list of other molecules.

Item 35. Diagnostic kits for items 1-34, the test analytes are IL-8, IL-1a, IL-1RA.

Item 36. It is a diagnostic kit of items 1-35, and further has a separation container having a buffer suitable for pre-wetting the sample pad of the separation cotton material 200.

Item 37. Diagnostic kits for items 1-36, the separate container having a running buffer.

Item 38. Diagnostic kits for items 1-37, the separate cotton material 200 is provided with a notch 206 on or near the edge of the end 204 of the support member 202, and the lateral flow device 300 is on the support member. The sample collection pads 201, 101 of the cotton material 200 form part of the porous support assembly 100 when the cotton material is inserted into the lateral flow device 300, having a curved portion 303 that fits snugly with the notch 206. ..

A method of detecting the presence or quantity of a test analyte of item 39.1 or higher.
a) A step of providing a separation cotton material 200 consisting of a sample collection pad 201 defined in any of the preceding items, the sample pad is a test sample taken from the skin surface of a mammal using the separation cotton material.
b) Inserting a cotton material consisting of a sample collection pad having the test sample into a lateral flow analyzer configured to accept the separated cotton material, as defined in one of the preceding items.
c) Steps to proceed with lateral flow analysis,
Consists of.

Item 40. The method of item 39, further comprising the step of adding a running buffer to the sample collection pad inserted into the lateral flow device.

Item 41. In the method of items 39-40, the lateral flow device comprises an elution zone 101 and a detection region DA. Here, the sample collection pad is the elution zone 101.

Item 42. The method of items 39-41, the skin surface of the mammal is human skin.

Item 43. The method of items 39-42, when the lateral flow device 300 is combined with a sample collection pad attached to cotton material, the lateral flow device is configured to form a porous support assembly 100. At that time, the lateral flow device 300 combined with the sample collection pad is composed of an elution zone 101, a junction zone 102, and a detection region DA.

Item 44. The method of items 39-43, the average thickness of the porous support assembly 100 is 4 mm or less, and the width and length are greater than the thickness. In this case, the lateral flow device is configured to substantially have a lateral flow direction L in the direction of the surface created by the width and length of the porous support assembly 100.

Item 45. Items 39-44, the detection region DA is a detection zone 105 containing one or more affinity molecules that selectively hold one or more test analytes, and optionally one or more indicator affinity molecules. Consists of an indicator zone 106 containing one or more affinity molecules that selectively retain.

Item 46. The methods of items 39-45, the test analytes are IL-8, IL-1a, IL-1RA.

Item 47. It is the method of items 39 to 46, and further has a step of d) capturing an image of the detection area DA and transmitting the image to a computer system including an image processing device and a database, and the image feature is an image processing device. The image feature is stored in a database, and the computer system generates at least one output data based on the image feature.

Item 48. Item 47, the image is captured using a mobile device, such as a mobile phone, which is configured to capture the image.

Item 49. Item 48, the output data generated by the computer system is transmitted to the mobile device.

Item 50. Items 39-49, the blocking buffer can be used to preprocess the sample collection pads 201, 101. Here, the blocking buffer is a PBS buffer composed of 1% BSA, or a buffer composed of 10 mM borate, 3% BSA, 1% PVP-40, 0.25% Triton X100 pH 8.0.

Item 51. The method of items 39-50, where the sample collection pads 201, 101 are prewet before the sample is taken.

"Example"
"Sample pad material and treatment"
"Example 1 In vitro test of sample pad material"
Different materials were tested as sample collection materials.
Samples were taken and in vitro tests of the best materials removed were performed. Pipette 80 μl of standard protein solution (PBS containing 2 ng / ml IL8, 4 ng / ml IL 1A, 8 ng / ml IL 1RA recombinant protein) onto parafilm and 1 x 1 cm different sample pad materials (block and non-block). It was absorbed using a block). Incubate the sample pad at room temperature for 5 minutes, insert into the SELF cassette, coat with FibroTx sample pad carrier (clear plastic piece, sample pad removed), close the cassette, 80 μl running buffer (PBS + 1%) Tween20) was applied and continued for 20 minutes, after which the results were read with a Qiagen ESEQuant LR3 lateral flow reader. The signal strength results are shown in mV.

The data is shown in FIG.

The unblocked sample pads had lower signal strength on the IL 1A test line compared to blocked sample pads of the same material. At the same time, sufficient IL 1RA removal failed and most of the sample pads tested were detectable. In IL 1RA, the blocked sample pad was detectable.

"Example 2 In vivo test of sample pad material"
The best material was selected based on in vitro results for in vivo testing. That is, C083, C048 (2 layers), 111 (2 layers), 222 (all blocks) were selected for testing on the skin. Also included was the first Fibro Tx sample pad (C095, unblocked).
A sample pad was placed on the skin and covered with a Fibro Tx sample pad bandage (sample pad removed). 90 μl of PBS was added and the sample pad was incubated on the skin for 15 minutes. The sample pad was inserted into a cassette (including pad carrier), 80 μl of running buffer (PBS + 1% Tween20) was added, continued for 20 minutes, and then the results were read with a Qiagen ESEQuant LR3 lateral flow reader. The signal strength results are shown in mV. The skin in the sun-exposed area (forehead) and the unexposed area (inside the arm) was tested in parallel.

FIG. 5A shows forehead data, and FIG. 5B shows medial arm data. On the skin, C083 showed strong signals on the forehead and medial arm. For this reason, this material was selected as the sample collection pad for the Fibro Tx SELF.

"Example 3 In vivo test block buffer"
Two different blocking buffers were tested for this material.
・ Simpler solution 1% BSA + PBS
-More complex solutions 10 mM Borate, 3% BSA, 1% PVP-40, 0.25% Triton-X100, pH 8.0

A blocked sample pad (C083) was tested on the skin inside the forearm and compared to a non-blocked sample pad.

The sample pad was placed on the skin and covered with a Fibro Tx sample pad bandage (sample pad removed). 90 μl of PBS was added and the sample pad was incubated on the skin for 15 minutes. The sample pad was inserted into a cassette (including pad carrier), 80 μl of running buffer (PBS + 1% Tween20) was added, continued for 20 minutes, and then the results were read with a Qiagen ESEQuant LR3 lateral flow reader. The signal strength results are shown in mV.

The C083 sample pad blocked with a more complex blocking buffer clearly shows the best results in skin tests using Fibro Tx SELF. The data is shown in FIG.

"Example 4 In vivo sampling procedure"
Biomaker samples can be obtained by incubating the sample pad on the skin or by wiping (wiping / rubbing) the sample pad on the skin. These methods are for comparing whether wiping can be used with the aim of simplifying customer sampling and reducing overall test time.
Samples taken by incubating the sample pad on the skin A sample pad (C083 block) is placed on the skin and covered with a Fibro Tx sample pad bandage. Add 2 drops of activation buffer (PBS) and leave the sample pad on the skin for 10 minutes.

Another sampling method-rub the sample pad on the skin:
Applicants were given the following directives: That is, two drops of PBS are added onto the sample pad, the pad with carrier is removed from the bandage, and the sample pad is rubbed on the skin in two different ways (both sides of the sample pad area are not touched).
a) Move the sample collection area to a Z shape and wipe it.
b) Move the sample collection area in a circular shape and wipe within the area of about 5 cm for 5 seconds.

After collecting the sample by any of the above methods, insert the sample pad into the cassette (including the pad carrier), add 2 drops of running buffer (PBS), leave for 20 minutes, and then Qiagen ESEQuant LR3 lateral flow reader. I read the result with. The signal strength results are shown in mV.

Forehead data is shown in FIG. 7A, and cheek data is shown in FIG. 7B.

The amount of sample (forehead) was too small for the Z-shaped method of wiping the skin.
For cosmetic purposes, we chose a method of wiping the sampling. This is because it saves the customer a lot of time and simplifies sampling.

We also tested long wipes.
Two drops of PBS were added to the sample pad and wiped on the skin by the following three methods.
-Move the sample collection area to a Z shape and wipe it.-Move the sample collection area to a circular shape and wipe it within an area of about 5 cm for 5 seconds.
-Move the sample collection area into a circular shape and wipe it within the area of about 5 cm for 30 seconds.

After collecting the sample, a centrifugal force is applied to the sample pad to elute it into the PBS having the collected sample. The biomaker level was determined using the enzyme-bound immunoadsorption assay. The biomaker level is shown in ng / ml.

The data is shown in FIG.
The 5-minute wipe yielded twice as much material from the skin as the Z-shaped transfer wipe. Longer time (30 seconds) wiping gave a much larger amount, but wiping for more than 10 seconds works because the sample pad material is crushed (depending on the strength and pressure of the wiping by the customer). Not suitable for testing.

"Example 5-In vivo test of skin treatment effect prior to test"
Analyze the effect of skin treatment / cleansing prior to the SELF test to determine the time required to apply a SELF bandage to the skin after the treatment.
Wipe the inside of the arm with a cotton pad moistened with mQ water / ethanol or moistened with a cosmetic wipe.

Subsequent treatments had control / untreated samples and averaged the three controls.

After this procedure, at various times, a sample pad (C083 block) was placed on the skin and covered with a Fibro Tx sample pad bandage. 80 μl of activation buffer (PBS) was added and the sample pad was incubated on the skin for 15 minutes. The sample pad was inserted into the cassette, 80 μl of running buffer (PBS) was added, continued for 20 minutes, and then the results were read with a Qiagen ESEQuant LR3 lateral flow reader. The signal strength results are shown in mV.

The data is shown in FIG.
a) Wash with 70% ethanol for maximum effect, primarily at IL 1RA levels
b) The right side of IL 1A and IL 1RA was slightly reduced with all treatments (IL 8 was not detected in healthy skin).
c) After 5 minutes, IL 1A and IL 1RA levels appear to have returned.
d) Prior to skin testing, customers can perform their usual skin care routines. That is, perform normal washing and apply normal cream / lotion / serum or the like.
e) I'm not saying that the 3 days before the skin test will not take extreme steps for the skin (unless it is required by the nature of the study) such as strong sunbathing, chemical / mechanical peeling, etc., but during the skin test the results will be You are advised not to apply too much oily cream / serum / lotion or heavy make-up (eg concealer, make-up cream, compact powder, etc.) as it may have an effect.

Claims (22)

1. 1. A diagnostic kit that detects the presence or absence of one or more test analytes in a test sample taken from the skin surface of a mammal, or detects the amount thereof.
a) Separation cotton materials 200, 301 configured to be used in collecting the test sample, the cotton material having sample collection pads 201, 101 attached to a support member 202.
b) Consists of a lateral flow analyzer 300, which is composed of one or more porosity elements and is configured to receive and hold the separated cotton material.
The sample collection pads 201, 101 are configured to form part of the porous support assembly 100 when the cotton materials 200, 301 are inserted into the lateral flow analyzer 300. A notch 206 is provided at one end of the end portion 204 of the support member 202, and the lateral flow device directs the end portion of the support member when the cotton material is inserted into the side flow device. The diagnostic kit according to claim 1, further comprising a curved portion 303 configured to be positioned. The separation cotton material 200 has a notch 206 on or near the end of the end 204 of the support member 202, and the lateral flow device 300 fits snugly with the notch 206 on the support member. It has a curved portion 303, which allows the sample collection pads 201, 101 of the cotton material 200 to form part of the porous support assembly 100 when inserted into the lateral flow device 300. The diagnostic kit according to claim 1 or 2, wherein the diagnostic kit is characterized. The end portion 204 of the support member 202 has an opening 205, the sample collection pads 201 and 101 adhere to the support member 202, and the sample collection pad covers the opening 205. The diagnostic kit according to any one of claims 1 to 3. The lateral flow device has an opening 304 configured to line up with the opening 205 of the cotton material, and when the cotton material is inserted into the side flow device, the sample collection pads 201, 101 The diagnostic kit according to claim 1 to 4, wherein the diagnostic kit is exposed through the opening 304. The diagnostic kit according to claim 1, wherein the support member 202 is flexible along the longitudinal axis of the support member. The support member 202 is made of a flexible material, and when the sample collection pads 201, 101 are pressed against the skin to collect a test sample and moved on the skin, the support member 202 is slightly lifted. The diagnostic kit according to claim 1-6, which is characterized by bending. The diagnostic kit according to claim 1, wherein the support member 202 is made of a plastic material, and the thickness of the plastic material is less than about 2 mm, such as less than 1 mm. 1. The support member has a near-end portion 203 configured as a finger grip, and the sample collection pads 201 and 101 are attached to the opposite end portion 204. Diagnostic kit according to 8. The sample pads 201 and 101 are made of a cellulosic material or a cellulose derivative such as nitrocellulose, polyether sulfone, polyethylene, nylon polyvinylidene fluoride (PVDF), polyester, polypropylene, glass fiber, cotton, cloth, etc., and are optional. The diagnostic kit according to claim 1 to 9, wherein the sample pads 201 and 101 are pretreated with a blocking buffer. Use blocking buffers such as PBS buffer consisting of 1% BSA or buffer consisting of 10 mM borate, 3% BSA, 1% PVP-40, 0.25% Triton X100 pH 8.0 for the sample collection pads 201 and 101. The diagnostic kit according to claim 1 to 10, wherein the diagnostic kit is pretreated. The diagnostic kit according to claim 1 to 11, wherein the sample collection pads 201 and 101 have the shape of a layer composed of one or more sheets, such as a layer composed of two sheets. The thickness of the sample collection pads 201 and 101 is less than 2 mm such as 1 to 0.80 mm, preferably less than 1 mm such as less than 0.95 mm, for example 0.85 mm or less such as 0.85 to 0.80 mm. The diagnostic kit according to claim 1-12. The sample pads 201 and 101 have the shape of a layer composed of two sheets, and the thickness of each sheet is less than 0.50 mm, such as 0.49 mm to 0.40 mm. The diagnostic kit according to 1 to 13. The lateral flow device is configured to receive the end portion of the cotton material so that the position of the sample collection pads 201, 101, 301 in the lateral flow device is ensured. The diagnostic kit according to claim 1 to 14, wherein the diagnostic kit comprises. The lateral flow device has a holding member 302 configured to hold the end of the cotton and ensure the position of the end of the cotton comprising the sample collection pads 201, 101. The claim 302 is characterized in that the holding member 302 is folded over the end of the cotton material and locked to the body of the lateral flow device, and the holding member 302 has the opening 304. Items 1 to 15 of the diagnostic kit. The lateral flow device is configured to form a porous support assembly 100 when coupled to the sample collection pad attached to the cotton material, and the lateral flow device 300 coupled to the sample collection pad It consists of an elution zone 101, a binding zone and detection region DA in the form of a binding pad 102, and optionally a suction pad 104, the detection region DA having one or more affinities that selectively hold one or more test analytes. To visually detect the detection zone 105 having sex molecules, the indicator zone 106 having one or more affinity molecules that optionally selectively retain one or more indicator affinity molecules, and the optional detection region DA. The diagnostic kit according to claim 1 to 16, wherein the reaction window 305 is configured in the above. A method of detecting the presence or quantity of one or more test analytes.
a) A step of providing a separate cotton material 200 consisting of a sample collection pad 201 as defined in any of the preceding claims, the sample pad being taken from the skin surface of a mammal, such as a human, using the separate cotton material. It is a test sample and
b) A cotton material consisting of a sample collection pad with said test sample is inserted into a lateral flow analyzer configured to accept the separated cotton material, as defined in any of the preceding claims, and optionally. A step of adding a running buffer to the sample collection pad inserted in the lateral flow device, and
c) Steps to proceed with lateral flow analysis,
Consists of. Sample collection pads 201, 101 are pretreated with blocking buffer, the blocking buffer being PBS buffer consisting of 1% BSA, or 10 mM borate, 3% BSA, 1% PVP-40, 0.25% Triton X100. The method of claim 18, wherein the buffer is made of pH 8.0. The method of claims 18-19, wherein the sample collection pads 201, 101 are pre-wet before collecting a sample. The method of claims 18-20, wherein the test analyte is IL-8, IL-1a, IL-1RA. Further, d) it has a step of capturing an image of the detection area DA and transmitting the image to a computer system including an image processing device and a database, extracting image features by the image processing device, and extracting the image features into a database. The computer system generates at least one output data based on this image feature and captures the image using a mobile device such as a mobile device configured to capture the image, as well as the computer system. The method according to claims 18 to 21, wherein the output data generated by the above-mentioned mobile device is transmitted to the mobile device.

Images